Ribosomal RNA methylation by GidB modulates discrimination of mischarged tRNA

Reviewer #1 (Public review):

Summary:

In this manuscript, Javid and colleagues worked to understand the molecular mechanisms involved in mistranslation in mycobacteria. They had previously discovered that mistranslation is an important mechanism underlying antibiotic tolerance in mycobacteria. Using a clever genetic screen they identify that deletion of gidB, a 16S ribosomal RNA methyltransferase, leads to lowered mistranslation (i.e. higher translational fidelity), but only in genetic backgrounds or environmental conditions that increase mistranslation rates.

Strengths:

The strengths of this manuscript are the clever genetic screen, the powerful mistranslation assays, and the clear writing and figures explaining a complex biological problem. Their identification of gidB as a factor important for mistranslation deepens our knowledge about this interesting phenomenon.

Weaknesses:

The structural work at the end feels like both an afterthought in terms of the science and the writing. I would suggest re-writing that section to be clearer about what the figure says and does not say. For example, the caption of Figure 6 appears to be more informative than the text and refers to concepts not present in the main text. In general, I found this section to be the most difficult to understand.

Reviewer #2 (Public review):

Summary:

Protein synthesis - translation - involves repeated recognition and incorporation of amino-acyl-tRNAs by the ribosome. This process is a trade-off between the rate and accuracy of selection (for review see (Johansson et al, 2008; Wohlgemuth et al, 2011)). The ribosome does not just maximise the rate or the accuracy, it balances the two. Therefore, it is possible to select mutants that translate faster than the wt (but are sloppy) or that are very accurate (more than the wt) but translate slower. Slow translation is detrimental as it limits the rate of protein synthesis (and, therefore, growth) and hyper-accurate mutants accumulate mis-translated proteins, which is detrimental for the cell.

Bi and colleagues employ genetics, MIC measurements, reporter assays, and structural biology to characterise the role of GidB rRNA methylase in translational accuracy in Mycobacterium smegmatis.

Strengths:

The genetics and phenotypic assays are convincing and establish the biological role of the methylase. The authors use a powerful set of complementary assays that convincingly demonstrate that the loss of GidB results in mistranslation.

Weaknesses:

(1) It would be essential to provide information regarding the growth rate and, ideally, translation rates in the gidB KO and the isogenic WT. As translation balances accuracy and speed, only characterising the speed is not sufficient to understand the phenomenon.

(2) Cryo-EM analysis of vacant 70S ribosomes is not sufficient for understanding the mechanisms underlying the accuracy defects in the gidB KO. One should assemble and solve structurally near-cognate and non-cognate complexes. I believe the authors are over-interpreting the scant structural data they have. Furthermore, current representation makes it impossible to assess the resolution of the structure, especially in the areas of interest.

References:

Johansson M, Lovmar M, Ehrenberg M (2008) Rate and accuracy of bacterial protein synthesis revisited. Curr Opin Microbiol 11: 141-147
Wohlgemuth I, Pohl C, Mittelstaet J, Konevega AL, Rodnina MV (2011) Evolutionary optimization of speed and accuracy of decoding on the ribosome. Philos Trans R Soc Lond B Biol Sci 366: 2979-2986.

Author response:

We thank the Dr. Ealand and Reviewers for their thoughtful comments on our submitted manuscript. We are in the process of revising our manuscript in light of the comments received, outlined below.

In addition to the requested revisions, we have new data with M. tuberculosis strain H37Rv +/- gidB deletion (and complementation), confirming that deletion of gidB sensitizes the strain to rifampicin, and extending our findings to pathogenic tuberculosis. This will also be incorporated into the revised manuscript.

Reviewer #1:

(1) The structural work at the end feels like both an afterthought in terms of the science and the writing. I would suggest re-writing that section to be clearer about what the figure says and does not say. For example, the caption of Figure 6 appears to be more informative than the text and refers to concepts not present in the main text. In general, I found this section to be the most difficult to understand.

We are rewriting this section to make it more coherent with the rest of the manuscript.

(2) "delta-gidB" is written out in the caption of Figure 6. Line 234: gidB not italics.

Thank you, these changes will be incorporated in the revised manuscript.

Reviewer #2:

(1) It would be essential to provide information regarding the growth rate and, ideally, translation rates in the gidB KO and the isogenic WT. As translation balances accuracy and speed, only characterising the speed is not sufficient to understand the phenomenon.

We are performing these assays and will incorporate them in the revised manuscript.

(2) Cryo-EM analysis of vacant 70S ribosomes is not sufficient for understanding the mechanisms underlying the accuracy defects in the gidB KO. One should assemble and solve structurally near-cognate and non-cognate complexes. I believe the authors are over-interpreting the scant structural data they have. Furthermore, current representation makes it impossible to assess the resolution of the structure, especially in the areas of interest.

While we agree with the Reviewer that structures of translating ribosomes will be most informative in elucidating the molecular mechanism(s) by which methylation (or not) by GidB contributes to mistranslation, those experiments are ongoing and beyond the scope of the current study. Unlike E. coli ribosomes, for which there are a plethora of structures for mutants available, there are very structures of mycobacterial ribosomes beyond wild-type apo ribosomes. Therefore we feel that the structures of apo mycobacterial ribosomes +/- GidB-mediated methylation are still of value, and a necessary “first step” for the mechanistic work alluded to above. Secondly, the apo ribosome structures still hint at potential mechanisms by which mistranslation and 16S rRNA methylation may impact on each other – as in the comments to R#1 above, we are revising the text to increase clarity and coherence of this section.